Bone morphogenetic proteins (BMPs) are a well-known family of growth factors that contribute to developmental processes such as pattern formation and tissue specification as well as promoting wound healing and repair processes in adult tissues. BMPs were initially isolated by their ability to induce bone and cartilage formation and are now known to regulate cell proliferation, migration, differentiation, and apoptosis in a number of tissues and organs.
BMPs include a number of related human proteins, such as BMP-2, BMP-3 (osteogenin), BMP-3b (GDF-10), BMP-4 (BMP-2b), BMP-5, BMP-6, BMP-7 (osteogenic protein-1 or OP-1), BMP-8 (OP-2), BMP-8B (OP-3), BMP-9 (GDF-2), BMP-10, BMP-11 (GDF-11, BMP-12 (GDF-7), BMP-13 (GDF-6, CDMP-2), BMP-15 (GDF-9), BMP-16, GDF-1, GDF-3, GDF-5 (CDMP-1), and GDF-8 (myostatin). BMPs may be grouped into subfamilies. For example, BMP-2 and BMP-4 are closely related, as are BMP-5, BMP-6, BMP-7, BMP-8, and BMP-8B. BMP-13, BMP-14, and BMP-12 also constitute a subfamily. BMPs are also present in other animal species. Furthermore, there is some allelic variation in BMP sequences among different members of the human population.
BMPs are a subset of the transforming growth factor-β (TGF-β) family, which also includes TGFs (TGF-β1, TGF-β2, and TGF-β3), activins (activin A) and inhibins, macrophage inhibitory cytokine-1 (MIC-1), Mullerian inhibiting substance, anti-Mullerian hormone, and glial cell line derived neurotrophic factor (GDNF). The TGF-β family is in turn a subset of the cysteine knot cytokine superfamily. Additional members of the cysteine knot cytokine superfamily include, but are not limited to, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), placenta growth factor (PIGF), noggin, neurotrophins (BDNF, NT3, NT4, and βNGF), gonadotropin, follitropin, lutropin, interleukin-17, and coagulogen.
BMPs have demonstrated utility in the treatment of a variety of conditions and diseases. BMP-2 and BMP-7 have been used to promote bone formation, bone fracture healing, and spinal fusion. BMP-4 (Rundle et. al., (2003) Bone 32: 591-601), BMP-5 (Arosarena and Collins (2003) Arch. Otolaryngol. Head Neck Surg. 129: 1125-1130), BMP-6 (Helm (2003) Gene Ther. 10: 1735-1743), and BMP-9 (Li et. al., (2003) J. Gene Med. 5: 748-756), have also been demonstrated to promote bone healing in animal models. Animal studies indicate that BMP-7 may be used to treat renal fibrosis and renal failure (Wang et. al., (2001) J. Am. Soc. Nephrol. 12: 2392-2399; Wang and Hirshberg (2003) Am. J. Physiol. Renal Physiol. 284: 1006-1013; Zeisberg et. al., (2003) Nat. Med. 9: 964-968; and Zeisberg et. al., (2003) Am. J. Physiol. Renal Physiol. 285: F1060-F1067), ischemic stroke (Chang et. al., (2003) Stroke 34: 558-564 and Harvey et. al., Pharmacol. Ther. (2005) 105: 113-125) and inflammatory bowel diseases (Maric et. al., (2003) J. Cell Physiol. 196: 258-264).
Structurally, BMPs are dimeric cysteine knot proteins. Each BMP monomer comprises multiple intramolecular disulfide bonds. An additional intermolecular disulfide bond mediates dimerization in most BMPs. BMPs may form homodimers; furthermore some BMPs may form heterodimers. BMPs are expressed as pro-proteins comprising a long pro-domain, one or more cleavage sites, and a mature domain. The pro-domain is believed to aid in the correct folding and processing of BMPs. Furthermore, in some but not all BMPs, the pro-domain may noncovalently bind the mature domain and may act as an inhibitor (e.g., Thies et. al., (2001) Growth Factors 18: 251-259).
BMP signal transduction is initiated when a BMP dimer binds two type I and two type II serine/threonine kinase receptors. Type I receptors include but are not limited to ALK-1, ALK-2 (also called ActRIa or ActRI), ALK-3 (also called BMPRIa), and ALK-6 (also called BMPRIb) and type II receptors include but are not limited to ActRIIa (also called ActRII), ActRIIb, and BMPRII. Following BMP binding, the type II receptors phosphorylate the type I receptors, the type I receptors phosphorylate members of the Smad family of transcription factors, and the Smads translocate to the nucleus and activate the expression of a number of genes.
BMPs also interact with inhibitors, soluble receptors, and decoy receptors, including BAMBI (BMP and activin membrane bound inhibitor), BMPER (BMP-binding endothelial cell precursor-derived regulator), Cerberus, cordin, cordin-like, Dan, Dante, follistatin, follistatin-related protein (FSRP), ectodin, gremlin, noggin, protein related to Dan and cerberus (PRDC), sclerostin, sclerostin-like, and uterine sensitization-associated gene-1 (USAG-1). Furthermore, BMPs may interact with co-receptors, for example BMP-2 and BMP-4 bind the co-receptor DRAGON (Samad et. al., (2005) J. Biol. Chem.), and extracellular matrix components such as heparin sulfate and heparin (Irie et. al., (2003) Biochem. Biophys. Res. Commun. 308: 858-865)
For further background on the BMP family, see Balemans and Hul (2002) Dev. Biol. 250: 231-250; Bubnoff and Cho (2001) Dev. Biol. 239: 1-14; Celeste et. al., (1990) Proc. Nat. Acad. Sci. USA 87: 9843-9847; and Cheng et. al., (2003) J. Bone Joint Surgery 85A: 1544-1552
A number of unfavorable properties of naturally occurring BMPs limit the development and use of BMP therapeutics. BMP expression yields are typically poor and suitable expression hosts are limited, hindering development and production. BMPs often possess multiple biological effects, including unwanted side effects. Many BMPs are poorly soluble, reducing storage stability and bioavailability. Finally, BMPs may induce unwanted immune responses.
Earlier studies have identified BMP variants with a number of interesting properties. BMP variants with improved yield in the context of E. coli expression and subsequent refolding from inclusion bodies have been disclosed (U.S. Pat. No. 5,399,677; U.S. Pat. No. 5,804,416; and U.S. Pat. No. 6,677,432). Consensus BMP variants with BMP-like activity have also been described (U.S. Pat. No. 5,011,691; U.S. Pat. No. 6,395,883; U.S. Pat. No. 6,531,445). A BMP-2 point mutant, L51 P, has been described that does not bind type I receptors but binds type II receptors normally (Keller et. al., (2004) Nat. Struct. Mol. Biol. 11: 481-488). Deletion mutants of BMP-4 that act as competitive inhibitors of BMP signaling have been disclosed (Weber et. al., (2003) J. Bone Miner. Res. 18: 2142-2151). Mutagenesis experiments have also been performed on BMP-2 to identify residues important for receptor binding; some of these variants were found to act as antagonists (Kirsch et. al., (2000) EMBO J. 19: 3314-3324 and Nickel et. al., (2001) J. Bone Joint Surg. Am. 83-A: S7-S14). In addition, methods for identifying analogs of morphogenetic proteins have been claimed (U.S. Pat. No. 6,273,598). Furthermore, several point mutants of ActA with reduced ALK-4 binding have been identified: S60P, I63P, M91E, I105E, and M108A (Harrison et. al., (2004) J. Biol. Chem. 279: 28036-28044).